Healthcare workers’ hands may harbor Staphylococcus spp. with antimicrobial resistance determinants in hospital settings, including pediatric care units. To characterize the antimicrobial susceptibility profile and detect the mecA gene in Staphylococcus spp. isolated from the hands of healthcare workers in maternal and pediatric hospital settings. Hand swab samples were collected from 60 healthcare workers, yielding 24 Staphylococcus isolates. Phenotypic identification, antimicrobial susceptibility testing by disk diffusion, and PCR detection of mecA were performed. Among the 24 isolates, 12 (50.0%) were coagulase-positive and 12 (50.0%) were coagulase-negative; no isolate was confirmed as Staphylococcus aureus. Resistance was highest to oxacillin (18/24; 75.0%), followed by clindamycin (15/24; 62.5%), gentamicin (14/24; 58.33%), sulfamethoxazole-trimethoprim (14/24; 58.33%), cefoxitin (14/24; 58.33%), and norfloxacin (13/24; 54.17%). Cefotaxime resistance was 50.0% and tetracycline resistance was 25.0%. The mecA gene was detected in 16 isolates (66.67%). Staphylococcus spp. recovered from healthcare workers’ hands showed substantial antimicrobial resistance, with mecA detected in two-thirds of isolates. These findings support continued surveillance and infection prevention measures in maternal and pediatric hospital settings.