Recent years have seen increasing interest in the construction of nanoscopically layered materials involving aqueous-based sequential assembly of polymers on solid substrates. In the booming research area of layer-by-layer (LbL) assembly of oppositely charged polymers, self-assembly driven by hydrogen bond formation emerges as a powerful technique. Hydrogen-bonded (HB) LbL materials open new opportunities for LbL films, which are more difficult to produce than their electrostatically assembled counterparts. Specifically, the new properties associated with HB assembly include: 1) the ease of producing films responsive to environmental pH at mild pH values, 2) numerous possibilities for converting HB films into single- or two-component ultrathin hydrogel materials, and 3) the inclusion of polymers with low glass transition temperatures (e.g., poly(ethylene oxide)) within ultrathin films. These properties can lead to new applications for HB LbL films, such as pH- and/or temperature-responsive drug delivery systems, materials with tunable mechanical properties, release films dissolvable under physiological conditions, and proton-exchange membranes for fuel cells. In this report, we discuss the recent developments in the synthesis of LbL materials based on HB assembly, the study of their structure–property relationships, and the prospective applications of HB LbL constructs in biotechnology and biomedicine.