The present study, which is part of a wider inter-disciplinary research project on Medieval Rome funded by the Swiss National Science Foundation, focuses on the archaeometric characterisation of glass tesserae from the apse mosaic of the church of S. Agnese fuori le mura, dated to the 7th century AD and never analysed until now. The main aims of the study are the identification of chemical compositions of glassy matrices and colouring/opacifying techniques by means of the combination of micro-textural, chemical, and mineralogical data. In S. Agnese tesserae, the results show the presence of glassy matrices and opacifiers/pigments, typical of both Roman and Late Antique/Early Medieval periods. The technological features identified (in particular, glassy matrices and opacifiers/pigments) allow us to discriminate not only new-production tesserae, i.e., those probably produced in the 7th century for the realisation of the S. Agnese mosaic, but also those obtained from recycling or re-using previous glass. This testifies to a quite complex “puzzle”, unusual in other glass mosaics from the same city and coeval with S. Agnese, supporting further the potentiality of archaeometric studies on glass to uncover the technical and socio-cultural knowledge that underpins its manufacturing, use, re-use, and recycling in the Early Medieval Rome.