The present study was planned to investigate the causative agent of Oreochromis niloticus summer mortality that resulted in significant economic losses in Port Said Governorate, Egypt during 2017.Many bacterial isolates were identified, among which Acinetobacter johnsonii has been selected to be the focus of the current study. Acinetobacter johnsonii_Egy was isolated and identified using the analytical profile index (API) 20 E, sequencing 16S ribossomal RNA gene fragment and phylogenetic analysis. A drug resistance evaluation revealed that A. johnsonii was resistant to ampicillin, gentamicin, lincomycin, nalidixic acid, tetracycline, and oxytetracycline among the tested antibiotics. Experimental infection was performed and challenged fish revealed similar clinical signs as seen in naturally infected ones. The disease appears to be highly infectious and lethal, causing 100% cumulative mortality during experimental infections. Virulence genes (fimH, traT, and iutA) and antibiotic resistance genes (qacED1, qnrS, sul1, dfrA, and aadA1) presence was confirmed in the A. johnsonii_Egy isolate. In conclusion, A. johnsonii is highly infectious to O. niloticus and might have a great influence on transfer of the antibiotic resistance in aquaculture. According to the available knowledge, this study is first to report A. johnsonii as emerging opportunistic pathogen in the Egyptian Nile tilapia (in Port Said).