Nitroaromatic compounds are toxic and rather recalcitrant pollutants of water and soil. Microbial degradation of the individual nitroaromatic compounds has already been well described in the literature. However, because several compounds often occur in the environment mutually influencing each other’s degradation, further research into the biodegradation of their mixtures is still needed. We investigated the degradation of a mixture of 4-nitrophenol (4-NP) and 4-nitrotoluene (4-NT) by a mixed microbial culture immobilized in a continuously operated packed-bed reactor (PBR) and by free suspended cells in shake flasks. Each compound was at first degraded separately, then in a mixture, and their degradation characteristics were compared. When treated separately, 4-NP and 4-NT were degraded with efficiency over 99 and 95%, respectively. When in a mixture, 4-NP was still completely removed from the media but the 4-NT removal efficiency dropped by a half and remained about the same during the whole experiment regardless the increasing 4-NP concentration at the PBR inlet. The shake flask experiments corroborated our finding that 4-NP negatively influenced 4-NT degradation by competitive inhibition and also indirectly on the adaptation level.