Scientific community is currently witnessing one of the most dynamic extinction risks, i.e. that of the largest bivalve mollusk of the Mediterranean, Pinna nobilis . The role of possible different pathogens responsible for the mass mortalities driving this extinction risk represent a considerable debate among researchers. After extensive investigation of the etiological agents, by means of numerous microbiological, pathophysiological, environmental studies, as well as efforts to identify surviving populations, current research is attempting to focus on the characterization of resistant genotypes that could enable future recruitment. Recently, the first characterization of the genomic architecture of Toll-like receptor (TLRs) genes in the genus Pinna identified potential SNPs associated with disease resistance and, hence, with genotypes tolerant to mortalities. Nevertheless, gene characterization is a challenging task that requires investigation at multiple levels, including gene expression under different stressors. In this context, the present study investigates TLR gene transcription in Pinna nobilis populations infected by different pathogens, comparing single infection with co-infection. In a general agreement with the proposed role of TLR4 and TLR6 which host SNPs associated with resistant or susceptible Pinna genotypes, the current results demonstrate a statistically significant increased transcription of these genes in individuals infected with both Mycobacterium sp. and Haplosporidium pinnae , the two mostly accused pathogens, compared with specimens infected with Mycobacterium sp. solely. Since analyses were only based on mantle tissue, these transcriptional associations require confirmation by multi-tissue and protein-level studies to clarify the functional role of TLRs in Pinna nobilis immunity and disease resistance. To this end, after examination of a large number, 11 primer pairs were validated in terms of efficiency and stability and are proposed for usage in other populations as well. • TLR expression was estimated in relation to different microbial load in Pinna nobilis. • The role of TLRs was confirmed in mantle of heavily infected individuals. • TLR4 and TLR6 hosting resistance associated SNPs demonstrated increased transcription. • Eleven TLR primer pairs were validated in terms of efficiency and stability.