© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing.Context: In recent years, microalgae have been used in the diet to improve the quality of animal products due to their oil content and quality, but determining the optimum dose is very important to prevent some fermentation and economic losses in animal production. Aims: The study investigated the effects of microalga (DHA-gold) supplementation in the diet at different levels in in vitro ruminal biohydrogenation and fermentation. Methods: The treatments were control (no additional oil, forage:concentrate ratio 60:40 on a dry-matter (DM) basis) or the addition of 1%, 2%, and 3% of microalga in the diet on a DM basis. Four rumen-cannulated goats were used as donors for the in vitro experiment. The treatments were incubated using a batch culture technique, and the fatty acid profile and fermentation parameters were determined after 0, 3, 6, 12, and 24 h. Key results: Total conjugated linoleic acids (CLA), cis-9, trans-11 CLA and trans-11 C18:1 fatty acids increased with supplementation of microalgae. The 24-h biohydrogenation rate of unsaturated fatty acids varied between 61% and 98%. In addition, the highest CLA content was obtained from the 3% algae group. Biohydrogenation of C18:3, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) fatty acids was decreased with the 2% microalga supplementation. The microalga supplementation had no effect on ruminal pH, ruminal acetate, propionate, and butyrate proportions, or the acetate:propionate ratio. Conclusion: Under the current in vitro study conditions, 2% microalga supplementation to diet increased the ruminal biohydrogenation intermediate products, such as total CLA and trans-11 C18:1 fatty acid and decreased the ruminal biohydrogenation ratio of n-3 fatty acids (C18:3, EPA, and DHA). Implications: Increasing the bioactive and beneficial fatty acids (e.g. CLAs) that are absorbed in the rumen by ruminants and then transferred to animal products (meat, milk) is important. From the results of this in vitro study, dietary microalga increased the ruminal CLA, C18:3, EPA, and DHA concentrations. However, dietary fat level should be evaluated in terms of improving an animal's performance (growth rate, digestibility, milk yield, carcass, and milk quality).