Previously, feeding fish oil (FO) and sunflower seeds to dairy cows resulted in the greatest increases in the concentrations of vaccenic acid (VA, t11 C18:1) and conjugated linoleic acid (CLA) in milk fat. The objective of this study was to evaluate the effects of forage level in diets containing FO and sunflower oil (SFO) on the production of trans C18:1 and CLA by mixed ruminal microbes. A dual-flow continuous culture system consisting of three fermenters was used in a 3 × 3 Latin-square design. Treatments consisted of (1) 75:25 forage:concentrate (HF); (2) 50:50 forage:concentrate (MF); and (3) 25:75 forage:concentrate (LF). FO and SFO were added to each diet at 1 and 2 g/100 g dry matter (DM), respectively. The forage source was alfalfa pellets. During 10-day incubations, fermenters were fed treatment diets three times daily (140 g/day, divided equally between three feedings) as TMR diet. Effluents from the last 3 days of incubation were collected and composited for analysis. The concentration of trans C18:1 (17.20, 26.60, and 36.08 mg/g DM overflow for HF, MF, and LF treatments, respectively) increased while CLA (2.53, 2.35, and 0.81 mg/g DM overflow) decreased in a linear manner ( P < 0.05) as dietary forage level decreased. As dietary forage levels decreased, the concentrations of t10 C18:1 (0.0, 10.5, 33.5 mg/g DM) in effluent increased ( P < 0.05) and t10c12 CLA (0.08, 0.12, 0.35 mg/g DM) tended to increases ( P < 0.09) linearly. The concentrations of VA (14.7, 13.9, 0.0 mg/g DM) and c9t11 CLA (1.78, 1.52, 0.03 mg/g DM) in effluent decreased in a linear manner ( P < 0.05) as dietary forage levels decreased. Decreasing dietary forage levels resulted in t10 C18:1 and t10c12 CLA replacing VA and c9t11 CLA, respectively, in fermenters fed FO and SFO.