The scope of practice of the osteopathic profession in Italy is underreported. The first part of the present study investigated the Italian osteopaths’ profile, focusing on the socio-demographic information and geographical distribution together with the main characteristics of their education. The OPERA-IT study highlighted that the majority of respondents declared to work as sole practitioners (58.4%), while the remaining declared to work as part of a team. Since teamwork and networking are recognized as fundamental aspects of health- care, the present study aims to compare the osteopathic practice, diagnostic and treatment modalities of osteopaths who work as a sole practitioner and osteopaths who work as part of a team to highlight possible differences. Moreover, patients’ characteristics will be presented. The OPERA-IT study population was chosen to provide a representative sample. A web campaign was set up to inform the Italian osteopaths before the beginning of the study. The OPERA IT study used a previously tested questionnaire. The questionnaire was translated into Italian following the World Health Organization recommendation. The questionnaire was composed of 57 items grouped in five sections, namely: socio-demographics, osteopathic education and training, working profile, organization, and management of the clinical practice and patient profile. The survey was delivered online through a dedicated platform. The survey was completed by 4,816 individuals. Osteopaths who work as sole practitioners represented the majority of the sample (n = 2814; 58.4%). Osteopaths who work as part of a team declared to collaborate mostly with physiotherapists (n = 1121; 23.3%), physicians with speciality (n = 1040; 21.6%), and other osteopaths (n = 943; 19.6%). The two groups showed heterogeneous characteristics. Significative differences were observed in all the factors, namely: geographical distribution, age, gender, training, working contract and working place, daily consultations and time for each consultation, fees, and the average waiting period to book an appointment. The principal component analysis supported a ten-component model and explained 80.5% of the total variance. The analysis showed that osteopaths working as sole practitioners have an increased probability (OR = 0.91; CI 95%: 0.88–0.94; p<0.01) of using systemic diagnostic and treatment techniques and have distinct clinical features with higher probability (OR = 0.92; 0.88–0.96; p<0.01) of spending less time with patients, being paid less but treating a higher number of patients per week. The most represented patients’ age groups were 41–64 years old (n = 4452; 92.4%) and 21–40 years old (n = 4291; 89.1%). Similarly, the most reported new patients’ age groups were 41–64 years old (n = 4221; 87.7%) and 21–40 years old (n = 3364; 69.9%). The most common presenting complaints were back pain, neck pain, cervical radiculopathy, sciatica, shoulder pain, and headaches. Osteopathic practice in Italy seems to be characterised by interprofessional collaboration, mostly with physiotherapists. Our results highlighted two different profiles in terms of sociodemographic characteristics and work modalities between osteopaths who work as sole practitioners and those who work as part of a team. Although according to the respondents, people of all ages consult Italian osteopaths, the majority of patients are adults. Most of them have been referred to osteopathy by other patients or acquaintances. Patients seek osteopathic care mostly for musculoskeletal related complaints.