Biomolecular assembly is a cornerstone of cellular organisation and function. Understanding its principles is essential for both elucidating biological processes and advancing therapeutic design. Multivalency, the cooperative binding of oligomeric subunits to form higher order assemblies, is a fundamental aspect of biomolecular interactions. Yet, it remains challenging to quantify, mainly due to the resultant molecular heterogeneity. Here, we present a mass photometry-based framework, enabling real-time, single-molecule visualisation of multivalent interactions in solution and on lipid membranes. We apply it to SARS-CoV-2 spike-ACE2 interactions and inhibition, as well as virus-like particle (VLP) assembly. We show that SARS-CoV-2 infectivity and inhibition correlates with cooperativity and oligomerisation rather than 1:1 binding affinities. ACE2 promotes spike oligomerisation in a variant-dependent manner, while antibodies exploit oligomerisation to enhance binding and inhibition. For VLPs, weak multivalent interactions drive hierarchical assembly through topologically stable intermediates consistent with the computed free energy landscape. Visualisation of individual assembly pathways events demonstrate that stochastic, state-dependent kinetics fine-tune the assembly process. Together, our results establish a general experimental platform for resolving biomolecular assembly and multivalent cooperativity controlling biological function with molecular resolution in real time.