The Pelagos Sanctuary is a marine biodiversity hotspot, as well as a busy zone for vessel traffic. Long term research on cetaceans has been important in guiding management of this protected area; however, non-cetacean species have received less attention. For this study, we included cetacean and non-cetacean species in an assessment of biodiversity and the potential for anthropogenic disturbance to cause ecosystem stress, indicated by a decline in species richness or evenness. We analyzed sightings of marine wildlife collected on whale watching vessels operating from Imperia and Genova, Italy, using 95% minimum convex polygons to quantify survey area in each region. Our analysis indicated similar levels of species richness in both areas, however Genova had lower Simpson and Shannon-Wiener diversity indices than Imperia. This suggests that Genova has lower species evenness, with the community being more dominated by common species, while rare species are more elusive. Disturbance was quantified based on vessel traffic and fishermen long lines. Genova exhibited significantly higher levels of vessel traffic, but there was no evidence of a difference in disturbance from long lines. These results suggest that anthropogenic disturbance in Genova may result in more ecosystem stress than Imperia. Consequently, management programs should prioritize reducing the impact of vessel traffic on marine life in the northwest Mediterranean Sea.