Background: Although guidelines recommend the use of chlorhexidine gluconate (CHG) for hand hygiene (HH), the impact of its routine use on antimicrobial resistance is not clear. Objective: To analyze the impact on the CHG susceptibility among isolates obtained from hands of HCW during its routine use for HH. Methods: We conducted a crossover study at 4 medical-surgical wards of a tertiary-care hospital in São Paulo, Brazil. In 2 units (intervention group), we established routine use of CHG for HH. For the other 2 units (control group), regular soap was provided. The availability of alcohol formulation for HH was not changed during the study. Every 4 months we swapped the units, ie, those using CHG changed for regular soap and vice versa. At baseline, we cultured the hands of HCWs. Only nursing staff hands were investigated. For hand culturing, HCWs placed their hands inside a sterile bag containing a solution of phosphate-buffered saline, Tween 80, and sodium thiosulfate. After the solution incubated overnight, it was inoculated onto brain-heart infusion. Next, it was plated on McConkey and Mannitol agar. MALDI-TOF was used for identification. Agar dilution was performed for Staphylococcus spp. We selected all Staphylococcus spp with MIC ≥ 8 and performed inhibition of efflux pump test. For isolates that showed a decrease of 2 dilutions, we searched the gene qacA/B by polymerase chain reaction. Results: We obtained 262 samples from HCW hands yielding 428 isolates. The most frequent genera were Staphylococcus spp (58%), Acinetobacter spp (8%), Enterobacter spp (8%), Stenotrophomonas spp (5%), Klebsiella spp (4%), Pseudomonas spp (3%), and others (14%). Staphylococcus spp were less frequent in the intervention compared to control group (43% vs 61%; OR, 0.48; 95% CI, 0.29–0.69; P = .005). Among all Staphylococcus spp, the proportion of chlorhexidine resistance (RCHG; MIC ≥ 8) was 12%. All resistant isolates recovered susceptibility after inoculation with pump-efflux inhibitor. For pump-inhibited isolates, 53% had the gene qacA/B amplified by PCR. We did not investigate RCHG among gram-negative isolates. There was a nonsignificant increase in Staphylococcus spp RCHG in the intervention group (4% to 6%; P = .90). Healthcare-acquired infection rates did not change significantly during the intervention. The consumption of CHG increased from 7.3 to 13.9 mL per patient day. Conclusions: We did not detect a significant difference in RCHG during the routine use of CHG for HH, although we observed increasing resistance. Further investigation is needed to clarify other reasons for increasing MIC to CHG.