The aim of this study was to evaluate the resistance to ten antimicrobial agents and the presence of bla ( TEM1 ) gene of Gram-negative bacteria isolated from three natural oligotrophic lakes with varying degrees of anthropogenic influence. A total of 272 indigenous bacteria were recovered on eosin methylene blue medium; they were characterized for antimicrobial resistance and identified taxonomically by homology search and phylogenetic comparisons. Based on 16S ribosomal RNA sequences analysis, 97% of the isolates were found to be Gram-negative bacteria; they belonged to 11 different genera. Members of the genera Acinetobacter, Enterobacter, and Pseudomonas predominated. Most of the bacteria were resistant to at least one antimicrobial. The incidence of resistance to beta-lactams, chloramphenicol, and mercury was high, whereas resistance to tetracycline, aminoglycosides, and nalidixic acid was low. There was a great frequency of multiple resistances among the isolates from the three lakes, although no significant differences were found among the disturbed and reference lakes. The ampicillin resistance mechanism of 71% of the isolates was due to the gene bla ( TEM1 ). Our study suggests that multiresistant Gram-negative bacteria and the bla ( TEM1 ) gene are common in freshwater oligotrophic lakes, which are subject to different levels of anthropogenic inputs.