This study evaluated the effects of lactic acid (LA) treatment of concentrates without or with inorganic P supplementation on ruminal fermentation profile and microbial abundances in non lactating cows. Six rumen-fistulated Holstein cows were assigned to a double 3 x 3 Latin square design with 3 experimental periods. Each period lasted 14 d, whereby the measurements were performed during the last 2 days. Cows were fed 3 diets containing untreated control concentrate supplemented with inorganic P, and two LA-treated concentrates, either without (LA - P) or with (LA + P) the inorganic P supplementation. The concentrate mixtures of the LA diets were soaked in 5% LA for 24 h before feeding, whereas the concentrate of control diet was not. All diets were offered as a total mixed ration (forage to concentrate ratio of 53:47). Ruminal pH, ammonia and short-chain fatty acid (SCFA) concentrations were determined in free ruminal liquid (FRL) and particle associated ruminal liquid (PARL) that was collected at 0, 2, 4, 8, and 12 h post-morning feeding. Target ruminal microbes in FRL and rumen solid digesta collected at 2 h post-feeding were analyzed using quantitative PCR. Cows consumed on average 17 +/- 1.0 kg DM/d (mean SEM), irrespective of the treatment. The concentration of total SCFA in FRT was increased by LA treatment (P < 0.001) without affecting the pH. Irrespective of P supplementation, feeding of LA-treated diets shifted SCFA profile towards more propionate in the FRL and PARL. The LA - P diet lowered ammonia concentration compared to the other diets (P < 0.05). The LA - P diet also reduced the fungal gene copies in solid digesta by 7.6% compared to the LA + P diet (P < 0.05). The total bacterial abundance in both ruminal fractions was not affected by diet; however, compared to the control, LA treatment enhanced (P < 0.05) the relative abundance of genus Prevotella in FRL (18%) and in solid digesta (27%). In contrast, the same treatment decreased the abundances of Clostridium cluster IV (23%) in FRL and Selenomonas ruminemtium group (30%) in solid digesta (P < 0.05). Abundances of fibrolytic microbes correlated with acetate and butyrate only in the rumen solid digesta. In conclusion, the LA treatment of concentrates caused major bacterial shifts, an increase of ruminal fermentation output, and enhancement of propionate fermentation without affecting ruminal pH. The lack of inorganic P supplementation did not impair rumen variables measured, but additional P supply and LA treatment of concentrates beneficially affected ruminal fungi. Lowered ruminal ammonia and branched-chain SCFA concentrations by the LA - P diet suggest lowered protein breakdown in the rumen by this treatment.