A novel method is described for simultaneous detection and quantification of attomoles or a few femtomoles of two (or potentially more) nucleic acid targets, without need for amplification. The technique depends on spectral–temporal resolution of chemiluminescence emitted from independent hybridization-induced chemiluminescent signal probes. The probes are internally quenched except in the presence of their specific targets, thereby allowing detection limits up to 10 000 times lower than with fluorescent probes. This is sufficient to obviate the need for amplification in many cases. The utility of the technique has been demonstrated by use of resolvable N-linked acridinium and 2,7-dimethoxyacridinium ester labeled probes in a homogeneous assay for sensitive and simultaneous independent quantification of pan-bacterial and pan-fungal target sequences in seawater.