Individual bacterial populations are known to respond differently toward substrate availability. To test how the availability of either pure phenol or natural humic matter (HM) selects for specific pelagic bacteria phylotypes from a humic lake (Lake Grosse Fuchskuhle, northeastern Germany), we used culture-dependent and -independent approaches. Using a batch approach, the bacterial community composition (BCC) differed depending on both the quantity and the quality of added substrates. Using a dilution-to-extinction approach, distinct BCC were detected by eliminating less abundant species. Most bacteria that were common in the lake were favored by phenol, and yet different subsets of the native BCC were enriched by HM. Specific bacterial groups with different growth requirements were consistently present, negatively influenced, or positively enriched following substrate additions. This study comprises the first explicit demonstration that bacteria such as Methylobacterium, Methylophilus, and Methylosinus spp. can be enriched on phenol or HM. Our isolation approaches led to the successful cultivation of a variety of native bacteria from the lake, such as Novosphingobium (Alphaproteobacteria) and Flexibacter (Bacteroidetes), or phenol-utilizing bacteria such as members of Actinobacteria or Burkholderia (Betaproteobacteria). Enrichment and cultivation on phenol and HM as substrates revealed highly specialized bacterial communities that resemble those found in many HM-rich lakes.