The administration of the anti-inflammatory medication sodium salicylate (SS) after calving has been shown to increase whole-lactation milk production in multiparous cows; however, treatment with SS is associated with hypoglycemia following its administration in some circumstances. We hypothesized that decreased glucogenic substrate supply from fermentation may contribute to decreased blood glucose concentrations in SS-treated cattle. We performed a 24-h batch culture experiment to determine the effects of SS on rumen microorganisms in vitro. Strained and pooled fluid from 3 heifers was combined in a 2:1 ratio with McDougall’s buffer, and 150 mL of the inoculum was added to each flask (n = 5/treatment). Blank flasks (n = 5) contained inoculum alone, while each treated and control flask contained 2.5 g of fermentation substrate. Before inoculum was added to the flasks, 1 mL of premixed treatment mixtures were added to achieve the desired final amount of SS (CON = 0 mg, LOW = 125 mg, MED = 250 mg, HI = 375 mg). Gas production was measured with the ANKOM^RF Gas Production System. Dry matter disappearance (DMD) was significantly depressed by inclusion of SS (P < 0.05), with HI having a lower DMD than LOW (P < 0.05), and MED intermediate (CON = 48%, LOW = 37%, MED = 30%, HI = 23% of DM; SEM = 2%). Final pH was not different between LOW and CON, but MED and HI had higher final pH than CON (CON = 6.31, LOW = 6.36, MED = 6.42, HI = 6.45; SEM = 0.01, P < 0.05). No differences were observed due to treatment for volume, rate, or lag in gas production (P ≥ 0.28). These results indicate that SS may have an inhibitory effect on rumen microorganisms in vitro, which is counterintuitive considering previous findings that SS administration in early lactation increases 305-d milk production, but consistent with previous observations of decreased blood glucose concentrations in SS-treated cows.