Mineral block is a strategic feed supplement for ruminants that provides a constant source of minerals to promote growth of rumen microbes. Hence, this study was conducted to investigate the effect of enhanced mineral blocks on in vitro rumen fermentation. Ruminal samples were collected from 3 ruminally cannulated Holstein Friesian cattle, and a commercial concentrate substrate was offered at 2g DM/100mL buffered ruminal fluid. Five enhanced mineral blocks were investigated and the treatments were: non addition (control), commercial mineral block (T1), T1+0.6ppm selenium (T2), T1+100ppm ammonium chloride (T3), T1+300ppm sodium bicarbonate (T4) and T1+0.03% molasses (T5). Each serum bottle was filled anaerobically and sealed before incubation at 0, 3, 6, 12 and 24 h. All treatments were conducted in triplicates and analyzed by ANOVA for randomized complete block design. Duncan’s multiple range test (DMRT) was used to identify differences among treatments and control. All analyses were carried out using SAS version 9.1 (SAS, 2002). All treatments resulted in decreased pH and increased gas production as incubation time increases. Total gas production was higher (P < 0.05) in T1 to T4 than that of T5 and control at 12 h of incubation. Ammonia-nitrogen (NH3-N) concentration was lowest (P < 0.05) in T4 (11.54 mM) after 24 h incubation followed by T5 and then T3. Also, propionate concentrations were higher (P < 0.05) in T2 to T5 at 12 h and T1 to T5 at 24 h incubation with T4 and T3 being the highest after 12 and 24 h of incubation with 22.56 mM and 27.55 mM, respectively. Acetate to propionate ratio was inversely proportional as the time increases in all treatment groups wherein lowest concentration was observed in T3 (1.47 mM) after 24 h incubation. Total volatile fatty acid (TVFA) was relatively higher in all treatments than the control group wherein T4 have the highest result after 12 and 24 h incubation with 72.13 and 86.56 mM, respectively. In conclusion, enhanced mineral block such as sodium bicarbonate (T4) can improve gas production, propionate and total volatile fatty acid for better rumen microbial fermentation.