Background: Empiric antibiotic therapy for suspected vertebral osteomyelitis (VO) should be initiated immediately in severely ill patients, and might be necessary for culture-negative VO. The current study aimed to identify differences between community-acquired (CA) and healthcare-associated (HA) VO in terms of clinical presentation, causative pathogens, and antibiotic susceptibility. Methods: Cases of adult patients with VO treated at a German university orthopaedic trauma center between 2000 and 2020 were retrospectively reviewed. Patient history was used to distinguish between CA and HA VO. Susceptibility of antibiotic regimens was assessed based on antibiograms of the isolated pathogens. Results: A total of 155 patients (with a male to female ratio of 1.3; and a mean age of 66.1 & PLUSMN; 12.4 years) with VO were identified. In 74 (47.7%) patients, infections were deemed healthcare-associated. The most frequently identified pathogens were Staphylococcus aureus (HAVO: 51.2%; CAVO: 46.8%), and Coagulase-negative Staphylococci (CoNS, HAVO: 31.7%; CAVO: 21.3%). Antibiograms of 45 patients (HAVO: n = 22; CAVO: n = 23) were evaluated. Significantly more methicillin-resistant isolates, mainly CoNS, were found in the HAVO cohort (27.3%). The highest rate of resistance was found for cefazolin (HAVO: 45.5%; CAVO: 26.1%). Significantly higher rates of resistances were seen in the HAVO cohort for mono-therapies with meropenem (36.4%), piperacillin-tazobactam (31.8%), ceftriaxone (27.3%), and co-amoxiclav (31.8%). The broadest antimicrobial coverage was achieved with either a combination of piperacillin-tazobactam + vancomycin (CAVO: 100.0%; HAVO: 90.9%) or meropenem + vancomycin (CAVO: 100.0%; HAVO: 95.5%). Conclusion: Healthcare association is common in VO. The susceptibility pattern of underlying pathogens differs from CAVO. When choosing an empiric antibiotic, combination therapy must be considered.