Edible Bird’s Nest (EBN) is a valuable and nutritious food tonic made from the saliva of Swiftlet birds. EBN has been consumed for centuries due to its high nutritional value. Proteins are the main nutrient in EBN. However, studies investigating the functional proteins in EBN are still limited. Therefore, the present study aims to characterize the functional proteins from EBN using proteomic-based techniques. In addition, the molecular characteristics of the functional proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that 0.951 ± 0.03 mg/mL of functional proteins were successfully extracted from the EBN. Several distinct protein bands in the range of 35–135 kDa were profiled from the EBN. Using ultrahigh performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS-MS), a total of 51 proteins were identified from the EBN. The protein sequences were processed in the BIOPEP database to predict the potential biological activity of EBN proteins. The prediction results suggest that the EBN proteins potentially possess antioxidant, anti-inflammatory, immunostimulatory, antitamnestic, antihypertensive (angiotensin converting enzyme (ACE) inhibitors), antidiabetic (alpha-glucosidase inhibitors, alpha-amylase inhibitors, dipeptidyl peptidase IV inhibitors) and antithrombotic properties. The antioxidant properties [DPPH scavenging activity (20.84% ± 3.43) and ABTS scavenging activity (31.49% ± 1.66)] and anti-inflammatory properties [inhibition of nitric oxide (NO) production (21.30% ± 4.41) and inhibition of albumin denaturation (25.30% ± 0.32)] were experimentally validated. In conclusion, the results suggest that functional proteins from EBN are potentially functional ingredients in the nutraceutical and food industries.