The use of antibiotics in animal production has been associated with the development and spread of antibiotic-resistant organisms including commensals. Coagulase-negative Staphylococcus (CoNS) species, which were until recently considered non-pathogenic, have been associated with opportunistic infections and high resistance to several antibiotics. This study sought to determine the prevalence, identity, and phenotypic resistance of coagulase-negative Staphylococcus spp. isolated from some selected poultry farms and farm workers in the Ashanti, Brong Ahafo, and Greater Accra regions of Ghana. Poultry litter samples and oral swabs of poultry farm workers were collected, from which bacterial species were isolated, identified, and analyzed. Various selective media were used for the presumptive identification of the different species. Confirmation of bacterial identity was done using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Antibiotic susceptibility testing of the isolates was performed using the Kirby-Bauer disk diffusion method. Zones of growth inhibition were interpreted based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Two hundred and fifty-six coagulase-negative Staphylococcus spp., comprising S. sciuri (42.97%), S. lentus (35.94%), S. gallinarum (6.64%), S. xylosus (4.30%), S. haemolyticus (3.91%), S. saprophyticus (1.95%), and S. cohnii (0.39%) were confirmed by MALDI-TOF. CoNS were isolated from samples from the Brong Ahafo (48.83%), Ashanti (33.59%), and Greater Accra (17.78%) regions. Isolates from poultry litter constituted 55.47%, and farm workers 44.53%. All the isolates were susceptible to amoxicillin/clavulanic acid and amikacin. The isolates exhibited high resistance toward tetracycline (57.03%), doxycycline (43.75%), and oxacillin (43.36%). Multi-drug resistance (MDR) was observed in 19.14% of the isolates. MDR was higher in isolates obtained from poultry farm workers (61.22%) than isolates from poultry litter (38.78%). The above findings call for stricter monitoring of antibiotic usage in both animal production and in humans.