The study was carried out to investigate the long-term (28 days) effects of different types and concentrations of C18 fatty acids (FA) on methanogenesis, fermentation, and microbial population in cultures of Entodinium caudatum (EC), Eudiplodinium maggii (EM), and Epidinium ecaudatum (EE). The ciliates were maintained in vitro on a basal ‘caudatum’ type culture medium, supplemented with a substrate mixture consisting of powdered meadow hay, wheat gluten (Sigma), crystalline cellulose (Sigmacell 20), and barley flour, together with an undefined prokaryotic population. Control cultures were maintained on the basal medium, whereas experimental ones were supplemented with stearic acid (SA), oleic acid (OA), linoleic acid (LN), and linolenic acid (LNA) at concentrations of 1, 5, 25, and 50 g/kg substrate mixture. After long-term cultivation, the following parameters were measured: pH, ammonia, short chain fatty acids (SCFA), methane concentration, and protozoal counts. Ciliates were enumerated under a light microscope, and methane level was determined by gas chromatography. Responses to fatty acids were species- and dose-dependent: EM was the most tolerant, whereas higher supplementation levels (≥25 g/kg) generally reduced protozoal density in EC and EE, with signs of gradual adaptation during long-term cultivation. Methane concentration decreased most markedly in EC at 50 g/kg, with reductions ranging from approx. 18 to 61% depending on the fatty acid. In EE, decreases occurred mainly under stearic acid supplementation and selected unsaturated fatty acid treatments, whereas total SCFA, pH and ammonia remained largely unchanged. Overall, C18 fatty acids can reduce in vitro methanogenesis, but the magnitude of the effect depends on protozoan species, fatty-acid type and dose.