The objective of this in vitro study was to examine the trans-10 (t10) shift in relation to the ratio between hydrogenating bacteria capable of producing either cis-9,trans-11 conjugated linoleic acid (c9t11 CLA) or t10,cis-12 CLA (t10c12 CLA). The influence of the in vitro condition on this shift was also investigated. Butyrivibrio fibrisolvens D1 (BF) and Propionibacterium acnes DSM 1897 (PA) were chosen as model organisms for the production of c9t11 CLA and t10c12 CLA, respectively. Different ratios of these bacteria (100/0, 50/50, 10/90, 2/98, 0.4/99.6, 0/100) were incubated in different growth media containing 40 µg/mL 18:2n-6 (LA): (1) control, (2) low pH, (3) 22:6n-3 (DHA) enriched media. The low pH medium was prepared by adding 2 M HCl to the control to reduce the pH from 6.5 to 5.5. The DHA enriched medium was the control supplemented with 40 µg/mL DHA. Under control conditions, the residual amount of LA after 24 h of incubation increased with increasing amounts of PA at inoculation (P = 0.013), which implies a lower rate of LA metabolism by the latter as compared with BF. Increasing amounts of PA also increased t10c12 CLA accumulation (P = 0.002) at the expense of c9t11 CLA (P = 0.006), with a t10 shift, defined as t10/t11 ≥ 1, occurring when PA represented between 90% and 98% of the inoculum. The required relative amount of PA at inoculation to induce a t10 shift decreased to 50% and 90% in the low pH or DHA enriched medium, respectively. Low pH or DHA addition did not stimulate t10c12 CLA formation, but inhibited CLA formation by both bacteria whereby PA seemed to be more tolerant. The current results suggest that besides a specific balance between BF and PA, specific external factors might influence the t11 to t10 shift. A low pH or, to a lesser extent, addition of DHA gives some advantage to PA compared with BF. Nevertheless, required proportions of PA remained high under all conditions. Hence, it is unlikely that PA is the only or predominant species involved in the t11 to t10 shift under in vivo circumstances.