Sesquiterpene lactones play various roles in plants, including allelopathy, antimicrobial activity, and protection against stressors. Higher concentrations of lactones are found in milky latex compared to leaves. Lactones contribute to the bitter taste of lettuce, balance human nutrition, and offer pharmaceutical benefits. This study aimed to examine the effect of biofertilisers on the sesquiterpene lactones content and overall taste of lettuce. Six Rijk Zwaan lettuce cultivars ('Kiribati', 'Murai', 'Aquino', 'Gaugin', 'Aleppo', and 'Carmesi') were grown in fertile soil using two biofertilisers (EM Aktiv and Vital Tricho), and a combination of the two during three successive growing seasons (autumn, winter, and spring). Preliminary UPLC-MS analysis of latex collected in autumn from all cultivars showed the presence of lactucin, deoxylactucin derivate, lactucopicrin, and lactucopicrin oxalate, with the lactucopicrin derivate being the prevailing form of sesquiterpene lactones. Concerning edible parts, the red cultivar 'Carmesi' exhibited the highest level of lactucopicrin (0.37 mg/g DW), with lactucopicrin being the predominant lactone in the autumn trial. Applying EM Aktiv and Vital Tricho alone resulted in significantly higher dihydrolactucopicrin content, while the combination of the two fertilisers led to significantly enhanced levels of all lactones. The results of the sensory analysis showed an overall taste score of 2.83-3.08 using a five-way hedonic scale. Cultivar and biofertiliser did not affect the overall taste, while a more pleasant taste was reported in winter/spring trials. Correlation coefficients revealed a negative impact of higher levels of lactucopicrin on overall taste (r=-0.16^*), suggesting a less bitter taste was associated with a lower concentration of this compound. Three-way ANOVA showed that the cultivar, fertiliser, and season jointly affected all of the tested parameters, indicating the need to consider multiple factors. Our findings are significant for applied and sustainable agriculture, showing that biofertilisers can affect lactones content and, at the same time, contribute to consumer acceptance of different cultivars regarding taste.