Enterococci are widely distributed bacteria incriminated in various clinical conditions in humans and economic losses in fish farms. The aim of this study was to recognize Enterococcus sp. and their virulence in the diseased Nile tilapia and humans and to assess the accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in recognizing Enterococcus species. Septicemic farmed O. niloticus (n=70) and human blood, urine, and pus samples (n=45) were collected from Sharkia Governorate and Suez Canal University Hospital, Ismailia, Egypt, respectively. Samples were bacteriologically and molecularly tested for the identification of Enterococcus sp.; moreover, the carriage of virulence genes by the isolates was evaluated. Proteomic (MALDI-TOF MS) was also used to assess its ability to identify Enterococcus sp. in comparison to the conventional PCR technique. Overall, Enterococcus sp. was identified in the Nile tilapia (32.9%) and human samples (28.9%). Two species of enterococci; E. faecalis and E. faecium, were identified. E. faecalis was the most prevalent species in both fish and human samples. High occurrence of four virulence genes (gelE) (86.1%), (cylA) (86.1%), (asa1) (77.8%), (esp < /em>) (72.2%) were detected in Enterococcus sp. isolates; however, (hyl) (19.4%) was recorded the lowest. Using MALDI-TOF MS, five of ten examined isolates were identified as Enterococcus sp.; however, all isolates were confirmed to be Enterococcus using conventional PCR. The study confirmed that PCR is the most accurate method for the identification of Enterococcus sp., while using MALDI-TOF MS as the only identification method is not advisable.