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Mortalities of <i>Pinna nobilis</i> populations set at risk the survival of the species from many Mediterranean coastline habitats. In many cases, both <i>Haplosporidium pinnae</i> and <i>Mycobacterium</i> spp. are implicated in mass mortalities of <i>P. nobilis</i> populations, leading the species into extinction. In the context of the importance of these pathogens’ role in <i>P. nobilis</i> mortalities, the present study investigated two Greek populations of the species hosting different microbial loads (one only <i>H. pinnae</i> and the second both pathogens) by the means of pathophysiological markers. More specifically, the populations from Kalloni Gulf (Lesvos Island) and from Maliakos Gulf (Fthiotis), seasonally sampled, were chosen based on the host pathogens in order to investigate physiological and immunological biomarkers to assess those pathogens’ roles. In order to determine if the haplosporidian parasite possesses a major role in the mortalities or if both pathogens are involved in these phenomena, a variety of biomarkers, including apoptosis, autophagy, inflammation and heat shock response were applied. The results indicated a decreased physiological performance of individuals hosting both pathogens in comparison with those hosting only <i>H. pinnae</i>. Our findings provide evidence for the synergistic role of those pathogens in the mortality events, which is also enhanced by the influence of seasonality.