Histone H2B contains a highly conserved C-terminal (H2B C) helix that has been implicated in chromatin interactions and dynamics. The H2B C helix is positioned adjacent to a major site of nucleosome interactions called the acidic patch. Despite individual structural studies highlighting interactions between chromatin proteins and the H2B C helix, the general role of the helix in mediating nucleosome recognition has not been explored. Moreover, many post-translational modifications (PTMs) have been identified within the H2B C helix, but significant gaps exist in our understanding of their regulatory potential. In this study, we employed nucleosome affinity proteomics using a library of nucleosomes with mutations or PTMs of the H2B C helix to investigate contributions to nucleosome binding. Our work uncovers new spatial patterns of H2BC helix engagement across the proteome. We also demonstrate that H2BK120 ubiquitylation (H2BK120ub1) broadly disrupts nucleosome binding, phenocopying mutation of the acidic patch, while differentially regulating acidic patch-dependent chromatin functions. In contrast, lysine acetylation results in more subtle position-specific changes, highlighting a more general role of H2B C helix PTMs in tuning acidic patch recognition.