Elevated levels of particulate matter (PM) in urban atmospheres are one of the major environmental challenges of the Anthropocene. To effectively lower those levels, identification and quantification of sources of PM is required. Biomonitoring methods are helpful tools to tackle this problem but have not been fully established yet. An example is the sampling and subsequent analysis of spider webs to whose adhesive surface dust particles can attach. For a methodical inspection, webs of orb-weaving spiders were sampled repeatedly from 2016 to 2018 at 22 locations in the city of Jena, Germany. Contents of Ag, Al, As, B, Ba, Ca, Cd, Co, Cr, Cs, Cu, Fe, K, La, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, S, Sb, Si, Sn, Sr, Th, Ti, V, Y, Zn and Zr were determined in the samples using inductively coupled plasma-mass spectrometry (ICP-MS) and inductively coupled plasma-optical emission spectroscopy (ICP-OES) after aqua regia digestion. Multivariate statistical methods were applied for a detailed evaluation. A combination of cluster analysis and principal component analysis allows for the clear identification of three main sources in the study area: brake wear from car traffic, abrasion of tram/train tracks and particles of geogenic origin. Quantitative source contributions reveal that high amounts of most of the metals are derived from a combination of brake wear and geogenic particles, the latter of which are likely resuspended by moving vehicles. This emphasizes the importance of non-exhaust particles connected to road traffic. Once a source identification has been performed for an area of interest, classification models can be applied to assess air quality for further samples from within the whole study area, offering a tool for air quality assessment. The general validity of this approach is demonstrated using samples from other locations.