Background and Aim: The emergence and rapid dissemination of multidrug-resistant (MDR) bacteria in different ecosystems is a growing concern to human health, animal health, and the environment in recent years. The study aimed to determine the antibiotic resistance in Escherichia coli from deer and nearby water sources at two different Safari parks in Bangladesh. Materials and Methods: A number of 55 fresh fecal samples of deer and six water samples from nearby lakes were collected from two Safari parks. Samples were processed, cultured, and carried out biochemical tests for E. coli. The antibiotic susceptibility was determined by disk diffusion method. To identify the resistance genes, polymerase chain reaction was performed. Results: A total of 32 E. coli isolates from 55 fecal samples and 6 of 6 E. coli isolates from lake water were isolated. From fecal E. coli isolates, ampicillin and sulfamethoxazole were 90.63% (n=29/32) resistant and 87.5% (n=28/32) were resistant to tetracycline and nalidixic acid. High resistance was also observed to other antibiotics. On the contrary, all E. coli isolates from water sources were 100% (n=6/6) resistant to ampicillin, tetracycline, sulfamethoxazole, and nalidixic acid. MDR was revealed in all water samples, whereas 96.88% (n=31/32) was found in fecal isolates. A number of blaTEM, tetA, and Sul2 genes were detected from both isolates. Conclusion: This study for the 1st time highlights, a significant proportion of E. coli isolates in wildlife deer and nearby water sources were MDR in Bangladesh.