This study evaluated bacterial co-cultivation as a strategy to mitigate brewer's spent grain (BSG) hemicellulosic hydrolysate toxicity, aiming to enhance 2,3-butanediol (2,3-BDO) and acetoin production through fermentation. Co-culture of Paenibacillus polymyxa with Pseudomonas alloputida or Rhodococcus sp. was assessed using synthetic medium and BSG hydrolysate. Attention was given to removing inhibitory compounds, including lignin-derived phenolics, hydroxymethylfurfural, furfural, and acetic acid, through microbial detoxification during co-cultivation. Various fermentation temperatures (30, 34, and 37 °C) and initial cell concentrations (OD600 of 0.05 and 0.1) were tested. Both P. polymyxa and Rhodococcus sp. effectively removed inhibitory compounds present in the medium. Co-cultures with Rhodococcus sp. exhibited higher sugar consumption rates (1.01 vs 0.88 g/L·h) than P. polymyxa monoculture, efficiently utilizing glucose, xylose, and arabinose, producing 2,3-BDO and acetoin. In co-culture with Rhodococcus sp., concentration (3.7 g/L), yield (0.14 g/g) and productivity (0.10 g/L·h) of 2,3-BDO at 34 °C considerably surpassed that of the P. polymyxa monoculture, with an increase of up to 48 %. These findings highlight the potential of co-cultures, especially with Rhodococcus sp., to alleviate inhibitory compound impacts when using complex media for fermentation. This study represents the first exploration of 2,3-BDO and acetoin production from BSG hemicellulosic hydrolysates using co-cultures.