Contamination with petroleum hydrocarbons causes extensive damage to ecological systems. On oil-contaminated sites, alkanes are major components; many indigenous bacteria can access and/or degrade alkanes. However, their ability to do so is affected by external properties of the soil, including nutrient cations. This study used Raman microspectroscopy to study how nutrient cations affect alkanes' bioavailability to Acinetobacter baylyi ADP1 (a known degrader). Treated with Na, K, Mg, and Ca at 10 mM, A. baylyi was exposed to seven n-alkanes (decane, dodecane, tetradecane, hexadecane, nonadecane, eicosane, and tetracosane) and one alkane mixture (mineral oil). Raman spectral analysis indicated that bioavailability of alkanes varied with carbon chain lengths, and additional cations altered the bacterial response to n-alkanes. Sodium significantly increased the bacterial affinity toward decane and dodecane, and K and Mg enhanced the bioavailability of tetradecane and hexadecane. In contrast, the bacterial response was inhibited by Ca for all alkanes. Similar results were observed in mineral oil exposure. Our study employed Raman spectral assay to offer a deep insight into how nutrient cations affect the bioavailability of alkanes, suggesting that nutrient cations can play a key role in influencing the harmful effects of hydrocarbons and could be optimized to enhance the bioremediation strategy.