The use of unconventional feedstuffs aims to incorporate alternative dietary options to contribute to the sustainability of livestock systems. The purpose of this study was to investigate the impact of incorporating Tithonia diversifolia (TD) into sugarcane (SC) silage on nutritional quality and ruminal fermentation in an in vitro gas production assay. The underlying hypothesis was that the increased protein content in silage with the addition of TD may result in a reduction in total gas production, including methane, due to potential modifications in the substrate fermentation profile. Treatments were formulated based on the inclusion of TD in SC silage, on a dry matter basis, as follows: TC: 100% SC; T25 = 75% SC + 25% TD; T50 = 50% SC + 50% TD; T75 = 25% SC + 75% TD. For the preparation of silages, forage samples were collected, subjected to pre-drying for approximately 24 h, then chopped, mixed according to each proposed treatment, and packed into mini silos (+/- 2 kg each) with increasing levels of TD. The silos were opened after 30 days, and samples were collected for chemical composition analysis and in vitro gas production assay. Silages with the addition of TD showed higher levels of crude protein and neutral detergent fiber compared to pure sugarcane silage. The addition of TD reduced total gas and methane production. The inclusion of Tithonia diversifolia in sugarcane silage demonstrated potential to enhance the nutritional quality of feed for ruminant production systems.