In the current study, biofilm forming ability and some virulence factors of 20 Yersinia ruckeri (Y. ruckeri) isolates obtained from rainbow trout (Oncorhyncus mykiss) were examined phenotypically. The Congo Red Agar (CRA), Modified Tube Aderans (Christensen) and Microplate methods were used to determine the biofilm forming ability of these isolates. The motility characteristics, hemolytic activity, lipase activity and protease activity of the isolates were determined by phenotypic tests. It was determined that the majority of the isolates produced a weak biofilm when examined using the microplate method, despite the fact that isolates did not produce biofilms when evaluated using the CRA and Christensen methods. While 14 isolates were discovered to have protease activity, 13 of the isolates were determined to be motile and to have lipase activity. All of the isolates were also determined to be nonhemolytic. According to the study’s results, a statistically significant, positive and strong correlation was found only between motility and lipase activity. The pathogenicity of microorganisms is associated with virulence factors and biofilm formation. Hence, we envisage that our results will significantly contribute to the establishment of novel techniques such as the development of anti-biofilm therapies for prevention or treatment of Y. ruckeri infections. We envisage that our results will significantly contribute to the development of new photonic and light sensing devices.