The main goal of our study was to find an optimal combination of tested factors to achieve lettuce rich in bioactive compounds sustaining its pleasant taste. We examined three red and three green cultivars in a greenhouse using two microbiological fertilisers (EM Aktiv and Vital Tricho), and their combination. Plants were grown in three consecutive growing seasons (autumn, winter, and spring). Lactones accumulated in autumn, whereas phenolics’ concentration rose during winter. Red cultivars showed higher phenolics and lactone content, where chicoric acid and luteolin-7-glucoside were the most abundant in the ‘Gaugin’ winter trial. Lactucopicrin was the predominant lactone among tested cultivars with the highest value in the red cultivar ‘Carmesi’. Solely applicated, the fertiliser EM Aktiv and Vital Tricho led to significantly higher phenolic acid and dihydrolactucopicrin content, while combined, there were notably increased levels of all detected lactones. Application of single fertilisers had no effect on flavonoid content, while the combination even reduced it. A sensory analysis showed a negative correlation between overall taste and total sesquiterpene lactones, lactucopicrin, caffeoylmalic, and chlorogenic acid, indicating a less bitter taste with decreasing content of these compounds. Our findings indicate that the cultivar, fertiliser, and growing season jointly affected all of the tested parameters, highlighting the differences in the application of EM Aktiv, Vital Tricho, and their combination.