Cardoon meal is a by-product of oil extraction from the seeds of Cynara cardunculus and can serve as a novel alternative feedstuff for ruminants. This study examined the rumen fermentation, biohydrogenation of fatty acids (FA) and microbial community in lambs fed a concentrate diet containing 15% dehydrated lucerne (CON, n = 8) or cardoon meal (CMD, n = 7) for 75 days pre-slaughter. Diets did not influence rumen fermentation characteristics and the abundance of bacteria, methanogens, fungi, or protozoa. Rumen digesta in CMD-fed lambs displayed a higher concentration of total saturated FA and lower total odd- and branched-chain FA and monounsaturated FA. Feeding CMD decreased total trans-18:1 isomer and the ratio of trans-10 to trans-11 C18:1, known as the “trans-10 shift”. Amplicon sequencing indicated that the rumen bacterial community in CMD-fed lambs had lower diversity and a higher relative phyla abundance of Proteobacteria at the expense of Bacteroidetes and Fibrobacteres. At the genus level, CMD mediated specific shifts from Prevotella, Alloprevotella, Solobacterium and Fibrobacter to Ruminobacter, suggesting that these genera may play important roles in biohydrogenation. Overall, these results demonstrate that cardoon meal can be used as a feedstuff for ruminants without negatively affecting rumen fermentation and microbiota but its impact on biohydrogenation may influence the FA composition in meat or milk.