Nursing staff have important roles to play in nosocomial infections. Antibiotic resistance is the ability of a microorganism to withstand the effects of an antibiotic; it evolves naturally via natural selection through random mutation, but it could also be engineered. The purpose of this study was to survey the relative frequency of antibiotic resistant strains in isolated bacteria from nursing staff. The present study was performed at one of the tertiary care hospitals in Isfahan, Iran, during a 24 month period (2007 to 2009). Eighty bacterial isolates from nursing staff were studied. Samples were collected with finger print method. Standard microbiological methods were performed for the detection of bacterial species. Bacterial antibiogram pattern according to Clinical and Laboratory Standards Institute (CLSI) 2010, were used by disk diffusion method. The result showed that Staphylococcus spp. 28 (53%), Bacillus spp. 48 (60%) and Enterobacteriaceae 4 (5%) were constituents of the isolated bacteria from nursing staff. According to the antibiogram pattern, 17.5, 59.5, 69.5, 41, 70.5, 50, 78, 97.5 and 64% of Staphylococcus spp.; 28, 30.5, 59, 65, 65, 77.5, 98, 93.5 and 100% of Bacillus spp. and 25, 73, 6, 75, 65, 50%, 64, 15 and 95% of Enterobacteriaceae were sensitive to Penicillin, Cephotaxime, Clindamycin, Co-trimoxazol, Ampicillin, Erythromycin, Tetracycline, Vancomycin and Gentamicin respectively. The results showed a high frequency of antibiotic resistant strains on nursing staff. Establishing systems for monitoring antimicrobial resistance in hospitals and the community and linking these findings to resistance and disease surveillance data is fundamental to develop treatment guidelines accurately and to assess the effectiveness of interventions appropriately.