Context Dietary manipulation is an important means to mitigate methane emissions; however, relatively few options exist for forage-based diets. Aims This batch culture study evaluated the effects of crude protein (CP) and fat concentration on ruminal DM disappearance (DMD), total gas (GP) and methane production, and ruminal fermentation in grass hay-based diets supplemented with a range of by-product feeds. Methods Eight treatments provided low or high CP (8 or 12% diet DM, respectively), with range of fat concentrations (1.8–6.0% diet DM), and included: control (grass hay only); corn distillers grains with solubles at 8% (CDDGS8) and 12% CP (CDDGS12); flax at 8% (FS8) and 12% CP (FSCDDGS12); canola meal at 8% (Can8); and sunflower screenings from Winkler (SFW8) or Deloraine (SFD8) at 8%. Data were analysed as a randomised complete block design, with fixed effect of treatment and random effects of block and treatment × block. Comparisons were performed between: (1) control and mean of by-product treatments, (2) low and high CP treatments, (3) CDDGS treatments, (4) FS treatments, and (5) SF treatments. Low- and high-fat treatments at both CP concentrations, and the means of CDDGS and FS treatments differing in fat concentrations were also compared. Key results The DMD did not differ between control and by-product treatments; however, DMD was lower (P < 0.001) in treatments with 8% compared with 12% CP, and was higher (P ≤ 0.009) with low fat compared with high fat, regardless of CP. Total GP was not affected by CP; however, at low CP, treatments with higher fat had lower GP (P ≤ 0.015). Methane production did not differ between any of the low and high CP treatments, but was higher (P ≤ 0.003) in the low-fat compared with high-fat treatments, regardless of CP, as well as in FS versus CDDGS and SFD versus SFW. Conclusions Higher CP increased DMD in vitro, but did not affect GP, methane or fermentation. Implications Higher dietary fat can mitigate enteric methane production, but can negatively impact DMD in grass hay-based diets, which is an important consideration in terms of animal performance.