Streptococcus iniae is one of the most hazardous fish pathogens infecting both farmed and wild fish species. The antibiotic resistance genes (ARGs) were inspected in Streptococcus iniae (S. iniae) isolates obtained from the cultured Nile tilapia, Oreochromis niloticus (O. nloticus), collected from fish farms in Kafr El-Shiekh Governorate, Egypt. The present study aimed to isolate and identify S. iniae and monitor the presence of some antimicrobial resistance genes. A total number of 30 Streptococcus isolates were recovered and then biochemically identified in the current study. Fifteen isolates (50%) were identified as S. iniae by polymerase chain reaction using 16S rRNA specific primers. The sensitivity of S. iniae isolates was recorded for erythromycin, tetracycline, norfloxacin, ciprofloxacin, oxytetracycline, streptomycin, sulfamethoxazole-trimethoprim, amoxicillin, and florfenicol. S. iniae showed high resistance to erythromycin and tetracycline. The tetracycline-resistant isolates contained the tet(M/O) gene, of which 3 and 2 isolates contained the tet(M) and tet(O) genes, respectively. Among the erythromycin-resistant isolates, 3 contained the erm(B) gene. The studied isolates showed a multiple antibiotic resistance profile, indicating difficulty in treating such infection. Thus, a need to develop a new strategy for controlling Streptococcus outbreaks in infected farms like vaccination is recommended.